The capabilities to introduce a particular foreign or native gene sequence into a mammal and to control the expression of that gene are of substantial value in the fields of medical and biological research. Such capabilities provide a means for studying gene regulation and for designing a therapeutic basis for the treatment of disease.
In addition to introducing the gene into mammals, providing expression of the gene at the site of interest remains a challenge. Methods have been developed to deliver DNA to target cells by capitalizing on indigenous cellular pathways of macromolecular transport. In this regard, gene transfer has been accomplished via the receptor-mediated encytosis pathway employing molecular conjugate vectors.
Inherited diseases of muscle pose a therapeutic challenge, as muscle is the single largest tissue of the body. Pharmacologic approaches do not significantly alter the course of many of these diseases as such approaches fail to correct the underlying genetic deficit. New approaches, relying on the transfer of genetic material have been advocated. However, current methodologies used for gene therapy are limited in their usefulness with regard to treating myopathies. Local administration of gene therapy vectors or transplantation of donor cells only treat the immediate area of the injection site. Effective therapy with these methods requires multiple injections and carry the safety risks associated with disease transplantation and the use of immunosuppressive drugs.
Duchenne muscular dystrophy is probably the most common inherited progressive lethal disorder of mankind. The rate of occurrence of the disease is attributable to the very high mutation rate of the gene. The progression of the clinical disease is characterized by skeletal muscle tissue deterioration and wasting. Duchenne muscular dystrophy (DMD) follows a degenerative course which confines sufferers to a wheelchair by the age of about 12 years old and results in death by the third decade due to cardiac or respiratory failure.
DMD results from mutations, mainly frame-shift deletions, in a single, recessively inherited gene. The gene has been cloned and represents the largest gene so far identified in the human genome, spanning at least 2.3 megabases in the short arm of the X chromosome.
Positional cloning of the X-linked gene has revealed that defects of the dystrophin gene lead to either Duchenne or Becker muscular dystrophy (BMD). The 14 kilobase (kb) dystrophin mRNA encodes a 3685 amino acid protein of 427 kilodaltons (kD) with overall similarity to the cytoskeletal proteins .beta.-spectrin and .alpha.-actinin. These proteins perform structural roles in static and dynamic cellular processes and all cell types. Dystrophin is associated with the cytoplasmic face of the sarcolemma and is thus an essential component of the muscle cytoskeleton. Although the exact function of dystrophin is unknown, it has been postulated to contribute to stabilization of the sarcolemma.
The current approaches to the in vivo transfer of dystrophin cDNA for the treatment of DMD have involved direct intramuscular injection of naked plasmid DNA or recombinant viral vectors. However, these techniques suffer from the limitation that skeletal muscle comprises a large proportion of the cells of the body, making widespread intramuscular transduction impractical in a clinical context. Furthermore, some muscles affected in DMD, such as the heart and diaphragm, are not readily accessable to direct injection.
Current approaches to gene therapy in inherited myopathies are limited in practicality either by the mode of administration or the capacity of the vector used for gene transfer. Therefore, methods are needed for targeting specific compositions to muscle cells.